Materials and methods for detecting the aryloxyalkanoate dioxygenase gene (AAD-12) containing event pDAB4472-1606 in plants

ABSTRACT

This application provides materials and methods for the detection of the aad-12 gene and event pDAB4472-1606 in biological samples derived from plants.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application Ser.No. 61/548,533, filed Oct. 18, 2011.

BACKGROUND OF THE INVENTION

Soybean is an important crop and is a primary food source in many areasof the world. The methods of biotechnology have been applied to soybeanfor improvement of agronomic traits and the quality of the product.Examples of agronomic traits introduced into soybean plants includeherbicide resistance and insect resistance.

The aad-12 gene (originally from Delftia acidovorans) encodes thearyloxyalkanoate dioxygenase (AAD-12) protein. This gene conferstolerance to 2,4-dichlorophenoxyacetic acid, for example, and topyridyloxyacetate herbicides. The aad-12 gene, itself, for introducingherbicide tolerance in plants was disclosed in WO 2007/053482.

The expression of heterologous or foreign genes (transgenes) in plantsis influenced by where the foreign gene is inserted in the chromosome.This could, for example, be due to chromatin structure (e.g.,heterochromatin) or the proximity of transcriptional regulation elements(e.g., enhancers) close to the integration site (Weising et al., Ann.Rev. Genet 22:421-477, 1988). Thus, the same gene in the same type oftransgenic plant (or other organism) can exhibit a wide variation inexpression level amongst different events. There may also be differencesin spatial or temporal patterns of expression. For example, differencesin the relative expression of a transgene in various plant tissues maynot correspond to the patterns expected from transcriptional regulatoryelements present in the gene construct introduced into the plant.

Thus, large numbers of events are often created and screened in order toidentify an event that expresses an introduced gene of interest to asatisfactory level for a given purpose. For commercial purposes, it iscommon to produce hundreds to thousands of different events and toscreen those events for a single event that has desired transgeneexpression levels and patterns. An event that has desired levels and/orpatterns of transgene expression is useful for introgressing thetransgene into other genetic backgrounds by sexual outcrossing usingconventional breeding methods. Progeny of such crosses maintain thetransgene expression characteristics of the original transformant. Thisstrategy is used to ensure reliable gene expression in a number ofvarieties that are well adapted to local growing conditions.

It would be advantageous to be able to detect the presence of atransgene and/or genomic DNA of a particular plant in order to determinewhether progeny of a sexual cross contain the transgene and/or genomicDNA of interest. In addition, a method for detecting the presence of thetransgene and/or genomic DNA in a particular plant would be helpful whencomplying with regulations requiring the pre-market approval andlabeling of foods derived from the recombinant crop plants.

It is possible to detect the presence of a transgene by any well knownnucleic acid detection method. Examples include the polymerase chainreaction (PCR) or DNA hybridization using nucleic acid probes. Thesedetection methods generally focus on frequently used genetic elements,such as promoters, terminators, marker genes, or other commonly usedgenetic elements. Such methods may not be useful for discriminatingbetween different events, particularly those produced using the same DNAconstruct unless the sequence of chromosomal DNA adjacent to theinserted DNA (“flanking DNA”) is known.

Event-specific PCR assays are, however useful in such a context andTaverniers et al. (J. Agric. Food Chem., 53: 3041-3052, 2005) disclosean event-specific tracing system for transgenic maize lines Bt11, Bt176,and GA21 and for canola event GT73. In Taverniers et al., event-specificprimers and probes were designed based upon the sequences of thegenome/transgene junctions for each event. Transgenic plant eventspecific DNA detection methods have also been described in U.S. Pat.Nos. 6,893,826; 6,825,400; 6,740,488; 6,733,974; 6,689,880; 6,900,014and 6,818,807.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to the AAD-12 soybean (Glycine max) eventdesignated pDAB4472-1606 having representative seed deposited withAmerican Type Culture Collection (ATCC) with Accession No. PTA-11028,and progeny derived thereof. The subject invention provides methods ofidentifying soybean plants having genomic DNA (gDNA) containing thisevent.

Thus, one aspect of the invention provides assays for detecting thepresence of the subject event in a biological sample (from soybeans, forexample). The assays can be based on the DNA sequence of the recombinantconstruct inserted into the soybean genome. Kits and conditions usefulin conducting the assays are also provided.

The subject invention also relates to nucleic acid sequences useful forthe cloning and analysis of the DNA sequences of a related to an aad-12insert. Certain embodiments of this aspect of the invention provideevent-specific primers for detecting an aad-12 insert and PCR ampliconsgenerated with these event-specific primer sets. Thus, these and otherrelated procedures can be used to uniquely identify soybean linescontaining an aad-12 insert (event pDAB4472-1606).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Scheme of a zygosity assay design. When the length of thetransgene is large (>5 kb), the Taq polymerase cannot amplify the largefragment with common and wild type specific primers. The result is thatonly the common and target specific primers amplify a DNA fragment,thereby only the transgene is detected.

FIG. 2. The assay involves performing a standard zygosity protocol astwo separate reactions which are outlined below: Reaction 1 (ControlReaction)—Common primer, Transgene specific primer, and a transgenespecific probe (FAM), and; Reaction 2 (Zygosity Test Reaction)—Commonprimer, Wild type specific primer, and a wild type specific probe (VIC).

FIG. 3. Standard zygosity assay calls.

FIG. 4. Distribution graph between sample numbers based on RelativeFluorescence Units (RFU). Null, homozygous and hemizygous spot groupingsare indicated within the circled areas.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the invention provides assays for detecting the presenceof transgenic soybean event pDAB4472-1606 in plant lines containing(comprising) these events as well as the cloning and analysis of the DNAsequences of the aad12 insert. Plant lines containing eventpDAB4472-1606 can be detected using primer and probe sequences disclosedherein.

The subject invention also provides assays and materials for detectingthe presence of event pDAB4472-1606 in order to determine whetherprogeny of a sexual cross between two soybean plants, at least one ofwhich contains the event of interest (event pDAB4472-1606). In addition,a method for detecting the event is helpful, for example, for complyingwith regulations requiring the pre-market approval and labeling of foodsderived from recombinant crop plants. As would be apparent to thoseskilled in the art, it is possible to detect the presence of eventpDAB4472-1606 by any well-known nucleic acid detection method such aspolymerase chain reaction (PCR) or DNA hybridization using nucleic acidprobes.

The introduction and integration of a transgene into a plant genomeinvolves random events (hence the name “event” for a given insertionthat is expressed) and, for many transformation techniques, such asAgrobacterium transformation, “gene gun” transformation and WHISKERS, itis unpredictable where a transgene will become inserted in the genome ofa plant.

At least 2500 seeds of a soybean line comprising event pDAB4472-1606have been deposited and made available to the public without restriction(but subject to patent rights), with the American Type CultureCollection (ATCC), 10801 University Boulevard, Manassas, Va., 20110. Thedeposit has been designated as ATCC Deposit No. PTA-11028. 100 packets(25 seeds per packet) of Glycine max seeds (AAD-12 Soybean EventpDAB4472-1606) were deposited on behalf of Dow AgroSciences LLC on Jun.10, 2010. The deposit was tested on Jun. 29, 2010, and on that date, theseeds were viable. This deposit was made and will be maintained inaccordance with and under the terms of the Budapest Treaty with respectto seed deposits for the purposes of patent procedure. The deposit willbe maintained without restriction at the ATCC depository, which is apublic depository, for a period of 30 years, or five years after themost recent request, or for the effective life of the patent, whicheveris longer, and will be replaced if it becomes nonviable during thatperiod.

Definitions and examples are provided herein to help describe thepresent invention and to guide those of ordinary skill in the art topractice the invention. Unless otherwise noted, terms are to beunderstood according to conventional usage by those of ordinary skill inthe relevant art.

A “biological sample” can comprise any organic material derived fromsoybean cells or tissue, including stems, roots, leaves, flowers orflower parts, seed or seed pods, and the like, that contains adetectable amount of a nucleotide sequence corresponding to such organicmaterial. A biological sample derived from soybean containing eventpDAB4472-1606 comprises the transgene/genome insertion regionscorresponding to event pDAB4472-1606.

A “probe” is an isolated nucleic acid to which is attached aconventional detectable label or reporter molecule, e.g., a radioactiveisotope, fluorophore, ligand, chemiluminescent agent, or enzyme. Such aprobe is complementary to a strand of a target nucleic acid, in the caseof the present invention, to a strand of genomic DNA containing eventpDAB4472-1606 whether from a soybean plant or from a sample thatincludes DNA from the event. Probes according to the present inventioninclude not only deoxyribonucleic or ribonucleic acids but alsopolyamides and other probe materials that bind specifically to a targetDNA sequence and such binding can be used to detect the presence of thattarget DNA sequence.

“Primers” are isolated nucleic acids that are annealed to acomplementary target DNA strand by nucleic acid hybridization to form ahybrid between the primer and the target DNA strand, and then extendedalong the target DNA strand by a polymerase, e.g., a DNA polymerase.Primer pairs of the present invention refer to their use foramplification of a target nucleic acid sequence, e.g., by the polymerasechain reaction (PCR) or other conventional nucleic acid amplificationmethods.

Probes and primers are generally 11 to 30 nucleotides or more in length.In certain instances, probes and primers can have lengths of more than30 nucleotides. Regardless of size, probes and primers can hybridizespecifically to a target sequence under high stringency hybridizationconditions. Preferably, probes and primers according to the presentinvention have complete sequence similarity with the target sequence,although probes differing from the target sequence and that retain theability to hybridize to target sequences may be designed by conventionalmethods.

As used herein, the term “progeny” denotes the offspring of anygeneration of a parent plant which comprises (or contains) aad-12soybean event pDAB4472-1606.

A transgenic “event” is produced by transformation of plant cells withheterologous DNA, i.e., a nucleic acid construct that includes atransgene of interest, regeneration of a population of plants resultingfrom the insertion of the transgene into the genome of the plant, andselection of a particular plant characterized by insertion into aparticular genome location. The term “event” refers to the originaltransformant and progeny of the transformant that include theheterologous DNA. The term “event” also refers to progeny produced by asexual outcross between the transformant and another variety thatincludes the genomic/transgene DNA. Even after repeated back-crossing toa recurrent parent, the inserted transgene DNA and flanking genomic DNA(genomic/transgene DNA) from the transformed parent is present in theprogeny of the cross at the same chromosomal location. The term “event”also refers to DNA from the original transformant and progeny thereofcomprising the inserted DNA and flanking genomic sequence immediatelyadjacent to the inserted DNA that would be expected to be transferred toa progeny that receives inserted DNA including the transgene of interestas the result of a sexual cross of one parental line that includes theinserted DNA (e.g., the original transformant and progeny resulting fromselfing) and a parental line that does not contain the inserted DNA.

As discussed above, one aspect of the invention relates to eventidentification. Related PCR primers and amplicons are included in theinvention. According to the subject invention, analytic PCR methodsusing amplicons that span inserted DNA can be used to detect or identifycommercialized transgenic soybean varieties or lines derived fromproprietary transgenic soybean lines containing event pDAB4472-1606.Thus, various embodiments of the invention provide primers comprisingSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 or various combinations thereofand probes comprising SEQ ID NO: 3, SEQ ID NO: 5 or both SEQ ID NOs: 3and 5.

Detection techniques disclosed herein are useful, in conjunction withplant breeding, to identify progeny plants containing eventpDAB4472-1606, after a parent plant containing said event is crossedwith another plant line in an effort to impart one or more additionaltraits of interest in the progeny. These PCR analysis methods benefitsoybean breeding programs as well as quality control, especially forcommercialized transgenic soybean seeds and can also benefit productregistration and product stewardship.

Thus, another aspect of the invention provides processes for makingcrosses using a plant containing event pDAB4472-1606 as at least oneparent (e.g., an F₁ hybrid plant having as one or both parents a plantcontaining event pDAB4472-1606) and analyzing the resultant progenyplants using primers and/or probes as disclosed herein. Aherbicide-tolerant soybean plant of the subject invention can be bred byfirst sexually crossing a first parental soybean plant consisting of asoybean plant grown from seed of the line referred to herein or from anysoybean plant containing event pDAB4472-1606, and a second parentalsoybean plant, thereby producing a plurality of first progeny plants;then selecting a first progeny plant that is resistant to a herbicide(or that possesses at least one of the events of the subject invention);selfing the first progeny plant, thereby producing a plurality of secondprogeny plants; and then selecting second progeny possessing eventpDAB4472-1606 using assays, primers and/or probes described herein.

One skilled in the art will also recognize that primers and probes canbe designed to hybridize, under a range of standard hybridization and/orPCR conditions, including conditions where the primer or probe is notperfectly complementary to the exemplified sequence. That is, somedegree of mismatch can be tolerated. For an approximately 20 nucleotideprimer, for example, typically one or two or so nucleotides do not needto hybridize with the opposite strand if the mismatched base is internalor on the end of the primer that is opposite the amplicon. Variousappropriate hybridization conditions are provided below. Furthermore,synthetic nucleotide analogs, such as inosine, can also be used inprobes. Peptide nucleic acid (PNA) probes, as well as DNA and RNAprobes, can also be used.

In some embodiments of the invention, compositions are provided fordetecting the presence of event pDAB4472-1606. These compositionscomprise event-specific primers, such SEQ ID NO: 2, and primers thathybridize with DNA common to the wild-type genome of plants that do notcontain event pDAB4472-1606 (SEQ ID NO: 1 and/or SEQ ID NO: 4). PCRanalysis demonstrated that soybean lines containing event pDAB4472-1606can be identified by analysis of the PCR amplicons generated with theseevent-specific primer sets (SEQ ID NO: 2). These and other relatedprocedures can be used to uniquely identify these soybean linescontaining event pDAB4472-1606. Thus, PCR amplicons derived from suchprimers and can be used to identify soybean lines containing eventpDAB4472-1606 and form another embodiment of the disclosed invention.

This invention also includes methods of detecting the presence of DNA,in a sample, that comprises plant material containing eventpDAB4472-1606. Such methods can comprise: (a) contacting the samplecomprising DNA with a primer set that, when used in a nucleic acidamplification reaction with DNA, produces an amplicon that identifiesevent pDAB4472-1606 within the sample (e.g., SEQ ID NOs: 1 and 2); (b)performing a nucleic acid amplification reaction, thereby producing theamplicon; and (c) detecting the amplicon.

Further detection methods of the subject invention include a method ofdetecting the presence of a DNA, in a sample, corresponding to eventpDAB4472-1606, wherein said method comprises: (a) contacting the samplecomprising DNA with a probe that hybridizes under stringenthybridization conditions with DNA from plant material containing eventpDAB4472-1606 and which does not hybridize under the stringenthybridization conditions with plant material from a control soybeanplant (a plant that does not contain event pDAB4472-1606, also referredto as non-event-of-interest DNA); (b) subjecting the sample and probe tostringent hybridization conditions; and (c) detecting hybridization ofthe probe to the DNA.

According to another aspect of the invention, methods of determining thezygosity of progeny of a cross with plants containing eventpDAB4472-1606 is provided. These methods can comprise contacting asample comprising soybean DNA with a primer set disclosed herein. Theseprimers, when used in a nucleic-acid amplification reaction with genomicDNA from a plant containing event pDAB4472-1606, produces a firstamplicon that identifies a plant as containing event pDAB4472-1606.These methods further comprise performing a nucleic acid amplificationreaction, thereby producing the first amplicon; detecting the firstamplicon; and contacting the sample comprising soybean DNA with anotherprimer set that, when used in a nucleic-acid amplification reaction withgenomic DNA from soybean plants, produces a second amplicon comprisingthe native soybean genomic DNA homologous to the soybean genomic region.The method can also further comprise performing a nucleic acidamplification reaction to produce a second amplicon, detection of thesecond amplicon and comparing the first and second amplicons in asample. The presence of both amplicons in a sample indicates that thesample is heterozygous for event pDAB4472-1606.

A “probe” is an isolated nucleic acid molecule to which is attached aconventional detectable label or reporter molecule (such as aradioactive isotope, ligand, chemiluminescent agent, or enzyme). Probesdisclosed herein include SEQ ID NOs: 3 and/or 5. Probes according to thepresent invention include not only deoxyribonucleic or ribonucleic acidsbut also polyamides and other probe materials that bind specifically toa target DNA sequence and can be used to detect the presence of thattarget DNA sequence. Methods for preparing and using probes and primersare described, for example, in Molecular Cloning: A Laboratory Manual,2nd ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

Depending on the application, one can use varying conditions ofhybridization to achieve varying degrees of selectivity of probe towardstarget sequence. For applications requiring high selectivity, one willtypically employ relatively stringent conditions to form the hybrids,e.g., one will select relatively low salt and/or high temperatureconditions, such as provided by about 0.02 M to about 0.15 M NaCl attemperatures of about 50° C. to about 70° C. Stringent conditions, forexample, could involve washing the hybridization filter at least twicewith high-stringency wash buffer (0.2×SSC, 0.1% SDS, 65° C.).Appropriate stringency conditions which promote DNA hybridization, forexample, 6.0× sodium chloride/sodium citrate (SSC) at about 45° C.,followed by a wash of 2.0×SSC at 50° C. are known to those skilled inthe art. For example, the salt concentration in the wash step can beselected from a low stringency of about 2.0×SSC at 50° C. to a highstringency of about 0.2×SSC at 50° C. In addition, the temperature inthe wash step can be increased from low stringency conditions at roomtemperature, about 22° C., to high stringency conditions at about 65° C.Both temperature and salt may be varied, or either the temperature orthe salt concentration may be held constant while the other variable ischanged. Such selective conditions tolerate little, if any, mismatchbetween the probe and the template or target strand. Detection of DNAsequences via hybridization is well-known to those of skill in the art,and the teachings of U.S. Pat. Nos. 4,965,188 and 5,176,995 areexemplary of the methods of hybridization analyses. In a particularembodiment, a primer or probe disclosed herein will specificallyhybridize to genomic DNA containing event pDAB4472-1606. Thehybridization of the probe or primer to DNA containing eventpDAB4472-1606 can be detected by any number of methods known to thoseskilled in the art, these can include, but are not limited to,fluorescent tags, radioactive tags, antibody based tags, andchemiluminescent tags.

Regarding the amplification of a target nucleic acid sequence (e.g., byPCR) using a particular amplification primer pair, “stringentconditions” are conditions that permit the primer pair to hybridize onlyto the target nucleic-acid sequence to which a primer having thecorresponding wild-type sequence (or its complement) would bind andpreferably to produce a unique amplification product, the amplicon. Theterm “specific for (a target sequence)” indicates that a probe or primerhybridizes under stringent hybridization conditions only to the targetsequence in a sample comprising the target sequence.

As used herein, “amplified DNA” or “amplicon” refers to the product ofnucleic-acid amplification of a target nucleic acid sequence that ispart of a nucleic acid template. For example, to determine whether thesoybean plant resulting from a sexual cross contains event pDAB4472-1606within its genomic DNA, DNA extracted from a soybean plant tissue samplemay be subjected to nucleic acid amplification method using a primerpair disclosed herein to produce an amplicon that identifies thepresence of event pDAB4472-1606. The amplicon may range in length fromthe combined length of the primer pairs plus one nucleotide base pair,and/or the combined length of the primer pairs plus about 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196,197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210,211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224,225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238,239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252,253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266,267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280,281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294,295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308,309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322,323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336,337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350,351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364,365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378,379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392,393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406,407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420,421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434,435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448,449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462,463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476,477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490,491, 492, 493, 494, 495, 496, 497, 498, 499, or 500, 750, 1000, 1250,1500, 1750, 2000, or more nucleotide base pairs (plus or minus any ofthe increments listed above). The use of the term “amplicon”specifically excludes primer dimers that may be formed in the DNAthermal amplification reaction.

Nucleic-acid amplification can be accomplished by any of the variousnucleic-acid amplification methods known in the art, including thepolymerase chain reaction (PCR). A variety of amplification methods areknown in the art and are described, inter alia, in U.S. Pat. No.4,683,195 and U.S. Pat. No. 4,683,202. PCR amplification methods havebeen developed to amplify up to 22 kb of genomic DNA. These methods aswell as other methods known in the art of DNA amplification may be usedin the practice of the present invention. The sequence of theheterologous transgene DNA insert from a subject soybean event can beverified (and corrected if necessary) by amplifying such sequences fromthe event using primers derived from the sequences provided hereinfollowed by standard DNA sequencing of the PCR amplicon or of the clonedDNA. The amplicon produced by these methods may be detected by aplurality of techniques. Agarose gel electrophoresis and staining withethidium bromide is a common well known method of detecting DNAamplicons.

TAQMAN (PE Applied Biosystems, Foster City, Calif.) is a method ofdetecting and quantifying the presence of a DNA sequence. Briefly, aFRET oligonucleotide probe is designed that hybridizes with a sequenceof interest (e.g., the aad12 gene or a gene native to the soybeangenome). The FRET probe and PCR primers are cycled in the presence of athermostable polymerase and dNTPs. During specific amplification, TaqDNA polymerase cleans and releases the fluorescent moiety away from thequenching moiety on the FRET probe. A fluorescent signal indicates thepresence of the sequence of interest due to successful amplification andhybridization. Molecular beacons have also been described for use insequence detection and can be used in accordance with the subjectinvention.

Thus, according to another aspect of the invention, methods of detectingthe presence of DNA corresponding to the event pDAB4472-1606 in abiological sample are provided. Such methods comprise: (a) contactingthe biological sample with a primer set that, when used in a nucleicacid amplification reaction with genomic DNA obtained from one or moresoybean plant(s) containing event pDAB4472-1606, produces an ampliconthat is identifies the plant as containing event pDAB4472-1606; (b)performing a nucleic acid amplification reaction, thereby producing theamplicon; and (c) detecting the amplicon wherein detection of saidamplicon is indicative of presence of the DNA corresponding to eventpDAB4472-1606.

According to another aspect of the invention, methods of detecting thepresence of a DNA corresponding to event pDAB4472-1606 in a biologicalsample are provided. These, methods comprise: (a) contacting thebiological sample with a probe that hybridizes under stringenthybridization conditions with genomic DNA from a soybean plantcontaining event pDAB4472-1606 and does not hybridize under thestringent hybridization conditions with a control soybean plant; (b)subjecting the biological sample and probe to stringent hybridizationconditions; and (c) detecting hybridization of the probe to the DNAcontaining event pDAB4472-1606, wherein detection of such hybridizationin indicative of presence of the DNA corresponding to eventpDAB4472-1606 within the genome of the plant. Preferably, the probe isSEQ ID NO:3.

Yet another aspect of the invention is a method of determining zygosityof the progeny of soybean plants containing event pDAB4472-1606. Themethod comprises (a) contacting a biological sample with a primer pairof SEQ ID NOs: 1 and 2, that when used in a nucleic acid amplificationreaction with genomic DNA from a biological sample containing genomicDNA from a biological sample from a soybean plant containing eventpDAB4472-1606, produces a first amplicon that identifies the presence ofthe aad-12 gene within the genomic DNA of the biological sample; (b)performing a nucleic acid amplification reaction; (c) detecting a firstamplicon produced (using SEQ ID NO: 3 as a labeled probe); (d)contacting the same sample with the primer pair SEQ ID NOs: 1 and 4,that when used in a nucleic acid amplification reaction with abiological sample containing genomic DNA from soybean plants produces anamplicon from the combination of primers that identifies wild-typesoybean genomic DNA homologous to the soybean genomic region of atransgene insertion identified as event pDAB4472-1606; (e) performing anucleic acid amplification reaction, and (f) detecting a second ampliconproduced (using SEQ ID NO: 5 as a detection probe); wherein: (a)detection of both amplicons indicates that the soybean sample isheterozygous for event pDAB4472-1606; (b) detection of only said firstamplicon (identifying the presence of the aad-12 gene) indicates thebiological sample is homozygous for event pDAB4472-1606; and (c)detection of only said second amplicon is indicative of the absence ofevent pDAB4472-1606 within the biological sample. The detection of theamplicons can be performed using labeled probes (e.g., SEQ ID NOs: 3 and5). As discussed above, probes can be labeled with a conventionaldetectable label or reporter molecule, e.g., a radioactive isotope,fluorescent label, ligand, chemiluminescent agent, or enzyme. In someembodiments, the probes are labeled with fluorescent labels that allowone to distinguish between an amplicon containing the aad-12 gene and anamplicon that does not contain the aad-12 gene (e.g.,fluorophore/quencher combinations such as 6-carboxyfluorescein (FAM) ortetrachlorofluorescin (TET) in combination with quenchers such astetramethylrhodamine, (TAMRA) or dihydrocyclopyrroloindole tripeptide(MGB).

In another embodiment of the invention, an aad-12 gene specific assay,such as that described in co-pending U.S. Provisional patent applicationSer. No. 61/548,543, filed on Oct. 18, 2011; MATERIALS AND METHODS FORDETECTING THE ARYLOXYALKANOATE DIOXYGENASE GENE (AAD-12) IN PLANTS;Inventors: Chandra Channabasavaradhya and Andrew Greenwald; herebyincorporated by reference in its entirety), can be paired with eventspecific PCR, such as that described and claimed herein. This embodimentcan be used to determine if there are mixed aad-12 events in the sameplant and whether the event detected by the event specific assay ishemizygous or homozygous. In this embodiment a gene specific assay isperformed and the gene copy number is estimated based on fluorescentsignal intensities produced by the gene specific assay. This is thencompared to that of a known event specific zygosity assay, such as thosedisclosed herein. If the aad-12 gene copy number estimation is ofsimilar intensity to that estimated by the event specific assay, then asample is deemed to have only the intended aad-12 event. If the copynumber estimated by the gene specific assay is more than that estimatedby the event specific assay, then the sample under study is deemed tohave additional aad-12 gene copies in addition to the intended event andhence indicates potential contamination of other aad-12 gene events.This scheme can also be used to detect contaminating events that bearidentical gene elements other than the aad-12 gene such as promoter orselectable marker sequences or any other identical sequences common toall the events. The latter approach can be applicable to determinezygosity and/or contamination of transgenic events of various traits buthaving similar elements in the gene cassette.

Kits for the detection of event pDAB4472-1606 in soybean plants andbiological samples derived therefrom are provided which use primersselected from SEQ ID NOs: 1, 2, and/or 4 and probes selected from SEQ IDNOs: 3 and 5. An amplicon produced using said kit is identifies abiological sample as containing event pDAB4472-1606 when the ampliconhybridizes with a probe comprising SEQ ID NO: 3. The kit can be providedas a means for specifically detecting the presence of eventpDAB4472-1606 within soybean plants (or biological samples derivedtherefrom) or the kit can be provided as a means for detecting amultiplicity of different transgenic events from any number of differentbiological samples. In the latter case, i.e., a kit for detecting amultiplicity of different transgenic events, the kit may provide probesor primers in the form of a micro array, or any sort of array whichprovides the user of said kit with the ability to distinguishdifferences between transgenic and non-transgenic samples, zygosity oftransgenic events, and even the presence or absence of events, whetherapproved or unapproved for commercialization. Detection or scoring ofthe presence or absence of certain events using such kits can be byfluorometric, colorimetric, isotopic, or chemiluminescent means.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety to the extent they are not inconsistent with theexplicit teachings of this specification.

The following examples are included to illustrate procedures forpracticing the invention and to demonstrate certain preferredembodiments of the invention. These examples should not be construed aslimiting. It should be appreciated by those of skill in the art that thetechniques disclosed in the following examples represent specificapproaches used to illustrate preferred modes for its practice. However,those of skill in the art should, in light of the present disclosure,appreciate that many changes can be made in these specific embodimentswhile still obtaining like or similar results without departing from thespirit and scope of the invention. Unless otherwise indicated, allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLES

An event specific TAQMAN ASSAY® was developed to detect the presence ofan MGB probe for event pDAB4472-1606 in soybean and to determinezygosity status of plants in breeding populations. To develop a genespecific assay, specific Taqman primers and probes were designedaccording to the DNA sequences located at the 5′ insertion site of eventpDAB4472-1606. For specific detection of event pDAB4472-1606 in soybean,a 209 by DNA fragment was amplified using two event specific primers.The amplification of this PCR product was measured by a target-specificMGB probe synthesized by Applied Biosystems containing the FAM reporterat its 5′end. Specificity of this Taqman detection method for eventpDAB4472-1606 in soybean was tested against event pDAB4472-1606 soybeanplants of varying levels of zygosity and non-transgenic soybean. Thesereactions were completed in a duplex format with wild type primersequences which amplified a 181 bp fragment which spans the insertionlocus of non-transgenic soybean (FIGS. 1, 2 and 3).

gDNA Isolation

Genomic DNA was extracted using the Qiagen DNeasy 96 Plant Kit. Freshsoybean leaf discs, 4 per sample, were used for gDNA extraction using amodified Qiagen DNeasy 96 Plant Kit protocol. The gDNA was quantifiedwith the Pico Green method according to vendor's instructions (MolecularProbes, Eugene, Oreg.). Samples were diluted with DNase-free waterresulting in a concentration of 10 ng/μL for the purpose of this study.

Taqman Assay and Results

Specific Taqman primers and probes were designed for the detection ofevent pDAB4472-1606 in soybean via a Taqman assay. These reagents can beused with the conditions listed below to detect event pDAB4472-1606within soybean. Table 1 lists the primer and probe sequences that weredeveloped specifically for the detection of event pDAB4472-1606 insoybean.

TABLE 1 PCR Primers and Probes Name Description 5′ to 3′ sequenceEvent Target Reaction D-Sb1606- Common  SEQ ID NO: 1 Cm-F ForwardGCCATTAATTATAGCGGTGTTTGC Primer D-SB1606- Reverse  SEQ ID NO: 2 Ev-RPrimer CGGTTAGGATCCGGTGAGTAATATT D-SB1606- Probe SEQ ID NO: 3 FMFAM-CCTTGCAAAATTC-MGB Reference System Reaction D-SB1606- Reverse SEQ ID NO: 4 Wt-R Primer GAATGAGGCTGCGTATAAAAATACAA D-SB1606- ProbeSEQ ID NO: 5 VC VIC-TGTGTTATTGGCCTCTTG-MGB

The multiplex PCR conditions for amplification listed in Table 2. Thecocktail was pipetted into a 96-well plate and was amplified using thefollowing conditions: i) 50° C. for 2 min., ii) 95° C. for 10 min., iii)95° C. for 15 sec, iv) 60° C. for 60 sec, iv) repeat step iii-iv for 35cycles, v) 4° C. hold. The Real time PCR was carried out on the BIO-RADICYCLER™ and ABI Gene Amp PCR System 9700 thermocylers. Data analysiswas based on measurement of the cycle threshold (Ct), which is the PCRcycle number when the fluorescence measurement reaches a set value. Ctvalue was calculated automatically by iCycler software.

Wild type control samples, which do not contain event pDAB4472-1606,will only produce Relative Fluorescence Unit (RFU) readings for the VICflurophore. Samples which are hemizygous or homozygous for eventpDAB4472-1606 genomic DNA will result in RFU readings for the FAMfluophore. These RFU readings are 0.5-1.0 units higher than that of thenegative background control. Samples yielding no PCR products for thetransgene or flanking genomic alleles may be the result of DNA that isnot of adequate quality or quantity, and is an indication that the assayshould be repeated. On completion of PCR and fluorescence readings, adistribution graph can be generated wherein samples which arehemizygous, homozygous or null are clustered as data points allowing thecut-off points to be visually determined.

TABLE 2 Reaction Mixture for event pDAB4472- 1606 specific Taqman insoybean. Component Concentration Volume TaqMan ® Genotyping Master Mix2x 2 μl Primer Mix 20 μM 0.1 μl D-Sb1606-VC (100 μM) 20 μM 0.04 μlD-Sb1606-FM (100 μM) 20 μM 0.04 μl PVP 0.05% 0.82 μl Sample DNA 1 μlTotal volume 4 μl

The Taqman detection method for event pDAB4472-1606 in soybean wastested against homozygous, hemizygous, and null samples in duplex formatwith soybean wildtype specific primers as a reference gene. This assayspecifically detected the event pDAB4472-1606 in soybean and did notproduce or amplify any false-positive results from the controls (seeFIG. 4). The event specific primers and probes can be used for thedetection of event pDAB4472-1606 in soybean and these conditions andreagents are applicable for zygosity assays.

We claim:
 1. A composition comprising a combination of primers andprobes, said combination of primers comprising SEQ ID NO: 1, SEQ ID NO:2 and SEQ ID NO: 4 and said combination of probes comprising SEQ ID NO:3 and SEQ ID NO: 5, said probes having a detectable label attached tosaid probe, said detectable label being selected from a radioactiveisotope, fluorescent label, chemiluminescent agent or enzyme and saidprobes and primers consisting of the recited SEQ ID NOS.
 2. Thecomposition according to claim 1, wherein said detectable label is aradioactive isotope.
 3. The composition according to claim 1, whereinsaid detectable label is a fluorescent label.
 4. The compositionaccording to claim 1, wherein said detectable label is achemiluminescent agent.
 5. A kit comprising a container and acomposition within said container, said composition comprising acombination of probes and primers, said combination of primerscomprising SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 4 and saidcombination of probes comprising SEQ ID NO: 3 and SEQ ID NO: 5, saidprobes having a detectable label attached to said probes, saiddetectable label being selected from a radioactive isotope, fluorescentlabel, chemiluminescent agent or enzyme and said probes and primersconsisting of the recited SEQ ID NOS.
 6. The kit according to claim 5,wherein said detectable label is a radioactive isotope.
 7. The kitaccording to claim 5, wherein said detectable label is a fluorescentlabel.
 8. A composition comprising a thermostable polymerase and acombination of nucleic acid sequences, the combination of nucleic acidscomprising a detectable label selected from a radioactive isotope,florescent label, chemiluminescent agent or enzyme, and the combinationof nucleic acid sequences consisting of the recited SEQ ID NO and beingselected from the following combinations of sequences: a) SEQ ID NO: 2and SEQ ID NO: 4; b) SEQ ID NO: 2 and SEQ ID NO: 5; c) SEQ ID NO: 3 andSEQ ID NO: 4; or d) SEQ ID NO: 3 and SEQ ED NO:
 5. 9. The compositionaccording to claim 8, wherein the detectable label is attached to SEQ IDNO 3 and SEQ ID NO
 5. 10. The composition according to claim 8, whereinsaid combination of nucleic acid sequences is SEQ ID NOs: 2 and
 4. 11.The composition according to claim 8, wherein said combination ofnucleic acid sequences is SEQ ID NOs: 2 and
 5. 12. The compositionaccording to claim 8, wherein said combination of nucleic acid sequencesis SEQ ID NOs: 3 and
 4. 13. The composition according to claim 8,wherein said combination of nucleic acid sequences is SEQ ID NOs: 3 and5.